Protein kinase C, a multigene family of phospholipiddependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase C␦, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase C␦ fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3-5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase C␦ also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase C␦-overexpressing cells. Stimulation of overexpressed protein kinase C⑀ did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.

Renal protein mass increases in diabetic renal hypertrophy. Accretion of protein may be the result of increased protein synthesis and/or decreased protein degradation. The lysosomal proteases, cathepsins B and L, are key enzymes in cellular protein catabolism. To evaluate the role of protein degradation in diabetic renal hypertrophy, the activities of cathepsins B and L were measured in microdissected proximal tubule segments and in kidney cortex homogenates. In rats four and ten days following induction of diabetes by streptozotocin, the kidney weight was increased and the cathepsin activities were reduced in proximal tubule segments. Treatment with insulin prevented both changes. The liver weight in diabetic rats was decreased and the activity of cathepsins B and L was increased, while the activity in kidney cortex was reduced. This excluded that diabetes per se may be accompanied by decreased cathepsin activities independent of organ hypertrophy. Renal hypertrophy as a cause rather than as the consequence of reduced cathepsin activities was excluded by the finding of unchanged cathepsin activities in proximal tubule segments from rats with compensatory renal hypertrophy four days and ten days following unilateral nephrectomy. Decreased activities of cathepsins B and L may reflect decreased intracellular protein degradation. Decreased protein breakdown in proximal tubules may contribute to diabetic renal hypertrophy. In agreement with this interpretation are the results from rats six months following induction of diabetes. Renal hypertrophy is complete at that time. No further accretion of protein occurs and the cathepsin activities in the proximal tubule were not different from controls.

Gentamicin accumulates in proximal tubule lysosomes, increases their number, and changes their structure. An important lysosomal function is degradation of intracellular proteins. To evaluate the effect of gentamicin on this lysosomal function, we measured the activity of the key lysosomal proteinases, cathepsin B and L, in microdissected S1, S2, and S3 segments of rat proximal tubules by means of a fluorometric microassay. The cathepsin activities were decreased in S1 and S2 following one and four gentamicin injections of 100 mg/kg body weight. The lysosomal enzyme, acid phosphatase, was also measured and was not decreased by gentamicin. The urine excretion of cathepsins B and L was decreased after gentamicin. This excludes an increase in urinary loss of cathepsins as the cause of decreased tubule activity. Structural changes of the lysosomes per se were excluded as the factor responsible for the reduced cathepsin activity by demonstrating increased cathepsin B and L activity in proximal tubule segments from rats injected with dextran, since dextran induces an increase in number and size of proximal tubule lysosomes. In vitro incubation of urine and tubule segments with gentamicin demonstrated a concentration-dependent reversible inhibition of cathepsin B and L. We conclude that gentamicin per se decreased cathepsin B and L activities in proximal tubule segments as early as 24 hours following one injection due to either enzyme inhibition or reduced generation of active intralysosomal cathepsin B and L. Gentamicin may, therefore, reduce renal protein catabolism by decreasing the activity of the key proteolytic enzymes, cathepsin B and L. Since cathepsin B and L are proteolytic activators of other lysosomal enzymes, their reduced activity may also decrease the activities of other lysosomal enzymes.

Renal extraction of low molecular weight proteins (LMWP) accounts for 30% to 80% of their total metabolic clearance. Extraction includes glomerular filtration, proximal tubular uptake, and intralysosomal proteolysis. To characterize the anatomic sites and enzymes involved in digestion of reabsorbed LMWP, the lysosomal proteases, cathepsin B and L, were measured by ultramicroassay in isolated S1, S2 and S3 segments of the proximal tubule of proteinuric rats. Increased glomerular filtration and tubular uptake of LMWP were induced by i.v. and i.p. injections of myoglobin and cationic and anionic lysozyme. Both cationic lysozyme and myoglobin increased cathepsin B and L activities in the proximal tubule, while anionic lysozyme had no effect. Morphologic examination of kidney tissue suggested that proximal tubular uptake of anionic lysozyme was negligible in comparison with the cationic form. Hence, only LMWP absorbed by the proximal tubule cells stimulated cathepsin B and L activities. Proximal tubular uptake of cationic lysozyme was determined by measurement of lysozyme activities in S1, S2, and S3. S1 segments contained the highest lysozyme activity, while S2 and S3 had much lower activities, and cathepsin B and L activity following cationic lysozyme injection was stimulated only in S1 segments. These results suggest that cathepsin B and L participate in lysosomal digestion of certain LMWP. Furthermore, the activities of cathepsin B and L adapt to increased uptake of LMWP. To gain additional insight into the mechanism of cathepsin adaptation, the cathepsin B and L activities were measured following injection of dextran with a similar low molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)